The scarcity of live human brain cells for experimental access has for a long time limited our ability to study complex human neurological disorders and elucidate basic neuroscientific mechanisms. A ...decade ago, the development of methods to reprogramme somatic human cells into induced pluripotent stem cells enabled the in vitro generation of a wide range of neural cells from virtually any human individual. The growth of methods to generate more robust and defined neural cell types through reprogramming and direct conversion into induced neurons has led to the establishment of various human reprogramming-based neural disease models.
Parkinson's disease (PD) is the second most prevalent neurological disorder and has been the focus of intense investigations to understand its etiology and progression, but it still lacks a cure. ...Modeling diseases of the central nervous system in vitro with human induced pluripotent stem cells (hiPSC) is still in its infancy but has the potential to expedite the discovery and validation of new treatments. Here, we discuss the interplay between genetic predispositions and midbrain neuronal impairments in people living with PD. We first summarize the prevalence of causal Parkinson's genes and risk factors reported in 74 epidemiological and genomic studies. We then present a meta-analysis of 385 hiPSC-derived neuronal lines from 67 recent independent original research articles, which point towards specific impairments in neurons from Parkinson's patients, within the context of genetic predispositions. Despite the heterogeneous nature of the disease, current iPSC models reveal converging molecular pathways underlying neurodegeneration in a range of familial and sporadic forms of Parkinson's disease. Altogether, consolidating our understanding of robust cellular phenotypes across genetic cohorts of Parkinson's patients may guide future personalized drug screens in preclinical research.
Patch-seq: Past, Present, and Future Lipovsek, Marcela; Bardy, Cedric; Cadwell, Cathryn R ...
The Journal of neuroscience,
02/2021, Volume:
41, Issue:
5
Journal Article
Peer reviewed
Open access
Single-cell transcriptomic approaches are revolutionizing neuroscience. Integrating this wealth of data with morphology and physiology, for the comprehensive study of neuronal biology, requires ...multiplexing gene expression data with complementary techniques. To meet this need, multiple groups in parallel have developed "Patch-seq," a modification of whole-cell patch-clamp protocols that enables mRNA sequencing of cell contents after electrophysiological recordings from individual neurons and morphologic reconstruction of the same cells. In this review, we first outline the critical technical developments that enabled robust Patch-seq experimental efforts and analytical solutions to interpret the rich multimodal data generated. We then review recent applications of Patch-seq that address novel and long-standing questions in neuroscience. These include the following: (1) targeted study of specific neuronal populations based on their anatomic location, functional properties, lineage, or a combination of these factors; (2) the compilation and integration of multimodal cell type atlases; and (3) the investigation of the molecular basis of morphologic and functional diversity. Finally, we highlight potential opportunities for further technical development and lines of research that may benefit from implementing the Patch-seq technique. As a multimodal approach at the intersection of molecular neurobiology and physiology, Patch-seq is uniquely positioned to directly link gene expression to brain function.
mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here, we report a neglected but important ...bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels, particularly when oxidative phosphorylation is impaired, such as in neurons treated with mitochondrial inhibitors, or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly, in mitochondrially defective neurons, but not neuroprogenitor cells, ribosomal S6 and S6 kinase phosphorylation increased over time, despite activation of AMPK, which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis, a major energy-consuming process, may account for its ATP-saving effect. We propose that a mild reduction in protein synthesis may have the potential to treat mitochondria-related neurodegeneration.
Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the ...essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.
Significance Neuronal cultures are very valuable to learn about basic principles of the nervous system. In vivo, neural electrical activity is the essence of nervous system function, controlling emotion, memory, sensory modalities, and behavior. In this study, we discovered that many crucial neurophysiological properties were strongly altered in classic culture media that are widely used by the research community. To overcome this problem, we designed and tested a new tissue culture neuromedium that adequately supports in vitro neuronal activity. The improvements made in this medium reduce the gap between in vivo brain physiological conditions and neuronal models in vitro. Improving physiological conditions in vitro may lead to more successful translation from bench to clinics.
Thousand of local interneurons reach the olfactory bulb of adult rodents every day, but the functional effect of this process remains elusive. By selectively expressing channelrhodopsin in ...postnatal-born mouse neurons, we found that their activation accelerated difficult odor discrimination learning and improved memory. This amelioration was seen when photoactivation occurred simultaneously with odor presentation, but not when odor delivery lagged by 500 ms. In addition, learning was facilitated when light flashes were delivered at 40 Hz, but not at 10 Hz. Both in vitro and in vivo electrophysiological recordings of mitral cells revealed that 40-Hz stimuli produced enhanced GABAergic inhibition compared with 10-Hz stimulation. Facilitation of learning occurred specifically when photoactivated neurons were generated during adulthood. Taken together, our results demonstrate an immediate causal relationship between the activity of adult-born neurons and the function of the olfactory bulb circuit.
Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may ...be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients.
The capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology are advancing. In parallel, cellular reprogramming and organoid engineering are expanding the ...use of human neuronal models in vitro. This creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identify multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (i.e., suboptimal fluorescence signals, phototoxicity, and unphysiological neuronal activity). To overcome these issues, we develop a neuromedium called BrainPhys™ Imaging (BPI) in which we optimize the concentrations of fluorescent and phototoxic compounds. BPI is based on the formulation of the original BrainPhys medium. We benchmark available neuronal media and show that BPI enhances fluorescence signals, reduces phototoxicity and optimally supports the electrical and synaptic activity of neurons in culture. We also show the superior capacity of BPI for optogenetics and calcium imaging of human neurons. Altogether, our study shows that BPI improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting optimal neuronal viability and function.
Despite widespread interest in using human induced pluripotent stem cells (hiPSCs) in neurological disease modeling, a suitable model system to study human neuronal connectivity is lacking. Here, we ...report a comprehensive and efficient differentiation paradigm for hiPSCs that generate multiple CA3 pyramidal neuron subtypes as detected by single-cell RNA sequencing (RNA-seq). This differentiation paradigm exhibits characteristics of neuronal network maturation, and rabies virus tracing revealed synaptic connections between stem cell-derived dentate gyrus (DG) and CA3 neurons in vitro recapitulating the neuronal connectivity within the hippocampus. Because hippocampal dysfunction has been implicated in schizophrenia, we applied DG and CA3 differentiation paradigms to schizophrenia-patient-derived hiPSCs. We detected reduced activity in DG-CA3 co-culture and deficits in spontaneous and evoked activity in CA3 neurons from schizophrenia-patient-derived hiPSCs. Our approach offers critical insights into the network activity aspects of schizophrenia and may serve as a promising tool for modeling diseases with hippocampal vulnerability.
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•Differentiation of human CA3 pyramidal neuron is modeled using ESCs/iPSCs•RNA-seq and immunocytochemistry revealed pyramidal neuron diversity•Rabies virus tracing showed a connection between ESC-derived DG and CA3 neurons•Schizophrenia hiPSC-derived DG-CA3 co-cultures present deficits in hippocampal activity
Sarkar et al. established a differentiation paradigm that generates human CA3 pyramidal neurons from ESCs and iPSCs and recapitulates hippocampal connectivity in vitro. This work reveals reduced levels of activity of schizophrenia-patient-derived neurons, offering opportunities for modeling diseases with hippocampal vulnerability.
Fluorizoline (FLZ) binds to prohibitin-1 and -2 (PHB1/2), which are pleiotropic scaffold proteins known to affect signaling pathways involved in several intracellular processes. However, it is not ...yet clear how FLZ exerts its effect. Here, we show that exposure of three different human cancer cell lines to FLZ increases the phosphorylation of key translation factors, particularly of initiation factor 2 (eIF2) and elongation factor 2 (eEF2), modifications that inhibit their activities. FLZ also impaired signaling through mTOR complex 1, which also regulates the translational machinery, e.g. through the eIF4E-binding protein 4E-BP1. In line with these findings, FLZ potently inhibited protein synthesis. We noted that the first phase of this inhibition involves very rapid eEF2 phosphorylation, which is catalyzed by a dedicated Ca2+-dependent protein kinase, eEF2 kinase (eEF2K). We also demonstrate that FLZ induces a swift and marked rise in intracellular Ca2+ levels, likely explaining the effects on eEF2. Disruption of normal Ca2+ homeostasis can also induce endoplasmic reticulum stress, and our results suggest that induction of this stress response contributes to the increased phosphorylation of eIF2, likely because of activation of the eIF2-modifying kinase PKR-like endoplasmic reticulum kinase (PERK). We show that FLZ induces cancer cell death and that this effect involves contributions from the phosphorylation of both eEF2 and eIF2. Our findings provide important new insights into the biological effects of FLZ and thus the roles of PHBs, specifically in regulating Ca2+ levels, cellular protein synthesis, and cell survival.
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